Immunoassays and assays based on the polymerase chain reaction (PCR) are among the most widely used techniques for detecting analytes. While PCR-based techniques generally give good detection sensitivity, they have not been widely adopted for routine screening in many laboratories. One reason is that when PCR is used for detecting analytes in biological materials, its accuracy is strongly influenced not only by the performance of the PCR assay itself, but also by the quality of the nucleic acids extracted from the biological materials being tested. In addition, the detection sensitivity of PCR-based methods can be reduced by inhibitors in the extract that may interfere with the amplification process. Trained lab personnel are also required to conduct PCR assays in order to assure their accuracy.
Immunoassays are generally less expensive and require less training to perform. They have been adopted in a variety of formats, with enzyme linked immunosorbent assays (ELISAs), lateral-flow immunoassays, and Western-blot assays being the most common. Current ELISA and Western-blot techniques, however, require multiple incubation steps and are prone to operator error. Lateral-flow immunoassays are generally faster but are not as accurate as conventional ELISAs.
An alternative assay format has been proposed by Tsien, et al. for detecting analytes. This technique, described in U.S. Pat. No. 5,998,204, makes use of a protein having an analyte-binding region and two fluorescent labels. When the analyte-binding region binds an analyte, a conformational change occurs which causes the two fluorescent labels to change position relative to each other. This alters a fluorescence resonance energy interaction between the labels, which is detected in order to determine analyte binding.
Frommer, et al. have proposed a similar assay format in PCT International Application No. 03/025220. This assay makes use of a fusion protein that includes a periplasmic binding protein portion and two fluorescent protein portions. The fusion protein changes conformation upon binding an analyte, changing the relative positions of the two fluorescent protein portions. An altered fluorescence resonance energy interaction is thereby induced between the fluorescent protein moieties.
The assay systems disclosed by Tsien and Frommer both require the use of sensor constructs which change conformation upon binding an analyte of interest. Both, moreover, detect only small analytes such as simple sugars and amino acids. There remains a need, therefore, for a more general approach to analyte detection which takes advantage of resonance energy transfer interactions but which is not limited to constructs that must change conformation in order to detect an analyte or that detect only small analytes.